Peptide binding by class II histocompatibility proteins is a critical event in the generation of adaptive immune responses and immunological tolerance. Previous studies supported by this project have demonstrated that the class II peptide loading pathway is highly regulated. HLA-DM plays a key role in the MHC class II presentation pathway; catalyzing peptide loading, editing the repertoire of peptides displayed to CD4+ T cells, and acting as a chaperone for empty class II molecules. However, the biochemical mechanisms underlying these functions are poorly understood. HLA-DO is a negative regulator of DM function, but its biological role remains to be defined. In this competing continuation application, experiments are proposed to further investigate factors that regulate peptide binding by MHC class II molecules with emphasis on delineating the mechanisms through which HLA-DM and DO regulate peptide exchange. In specific aim 1, biochemical experiments are proposed to further investigate the structural basis for DM function and to test the hypothesis that DM is subject to allosteric regulation. Additional experiments are proposed to analyze the interaction of class II molecules with tetraspanin proteins, and to test the hypothesis that tetraspanins regulate peptide loading by controlling the interaction of DM with substrate class II molecules. The second aim focuses on HLA-DO, including a biochemical analysis of how DO regulates DM function. In addition, experiments are proposed to test the hypothesis that the primary function of H2-O is to broaden the array of self-peptides presented by tolerogenic APCs, through attenuation of DM-mediated peptide editing. These studies are expected to advance our understanding of the mechanisms that regulate peptide loading in the class II antigen presentation pathway, which plays a fundamental role in determining the specificity of immune responses and immunological tolerance. This work will therefore impact our understanding of the pathogenesis of autoimmune diseases such as lupus and diabetes, with the potential to provide new avenues for therapeutic manipulation of immunity and tolerance.